The present invention relates generally to the field of chemical analysis. More specifically, the invention relates to a method for electrochemical detection at low potentials.
Chemical analysis is widely used in modem society to examine samples obtained from a variety of sources to determine the presence or absence of analytes of interest. Examples include: drug detection in human and animal tissue and secretions; detection of contamination in water, food and the environment; scientific research; and medical diagnosis. In the food industry, for example, chemical analysis is routinely used to analyze a large number of tissue samples from food-producing animals. In this way, drug residue levels can be determined for food safety purposes. Regardless of the application, techniques for chemical analysis should be rapid, performable by laboratory technicians, inexpensive, and sensitive.
Currently, high performance liquid chromatography (HPLC) is the most common method of separating analytes for analysis. Although HPLC provides good selectivity for this application, it has several drawbacks. The most significant of these is its high cost of operation. HPLC uses high-cost equipment and consumables. In addition to these costs is the increasing cost of disposing of the hazardous solvent waste generated by HPLC. Capillary electrophoresis (CE), first reported in the early nineteen eighties, has developed into a technique with a substantial application base and commercial support. Because CE does not suffer from the same high cost of operation as HPLC, it would seem to be a good replacement.
Analytical methods such as Flow Injection Analysis (FIA) use selective chemical transformation of an analyte, rather than separation, to enable one analyte to be detected or quantified in the presence of another. The chemical modification is carefully selected to allow the desired substance(s) to register on a suitable detector, while components of the mixture that are not modified do not register.
Once the constituents of a fluid are separated by CE or another suitable separation scheme, or are chemically modified by FIA or similar method, they must be analyzed by a detection system that identifies and preferably also quantifies the level of each separated or modified constituent. Commercially available detection methods for use in conjunction with CE separation do not provide particularly high sensitivity.
UV detection has been the most popular detection system available for use in conjunction with CE separation. UV detection is a mass-dependent detection method, however, and its sensitivity is severely compromised by the short light path lengths encountered in CE. Laser-induced fluorescence has been used to achieve sensitive detection but is limited to compounds which fluoresce or are amenable to being tagged with a fluorophore.
Another method of detection is amperometric electrochemical detection. See, e.g., Wallingford et al., 1987 Anal. Chem. 59: 1762-1766. Wallingford et al. describe an amperometric electrochemical detection (ECD) for CE with a carbon fiber electrode inserted into a detection capillary. The detection capillary, in turn, is butted up to the CE separation capillary. The separation and detection capillaries are joined using an ion-conductive material through which the separation current could flow. Using this configuration, the ECD system is effectively decoupled from the CE separation field. When using a carbon fiber electrode inserted into the end of the capillary, a decoupling system is employed to avoid a high voltage drop along the length of the fiber electrode within the capillary, parallel to the separation field. A large number of amperometric-based CE-ECD reports followed with improved decoupling systems.
Despite these improvements, such field-decoupling systems are difficult to construct, fragile and impractical for many applications. Disk electrode systems, which do not require field decoupling systems, employ a disk electrode at the end of the separation capillary, normal to the separation field. Although the potential of the sensing electrode is shifted by the separation field, this potential shift is constant across the electrode surface and can therefore be accounted for by including an xe2x80x9coffsetxe2x80x9d in the potential applied to the sensing electrode. Unfortunately, disk-electrode systems typically demonstrate sensitivities significantly lower than those of the more complex carbon-fiber electrode, field-decoupled CE-ECD systems.
Amperometric ECD detection has proven a fairly sensitive ECD mode used for CE. Other ECD modes, such as conductionmetric and potentiometric detection, are typically less sensitive and apply only to a narrow group of analytes. Voltammetric ECD is also generally less sensitive than amperometric detection. In conjunction with HPLC and CE separation systems, however, lower sensitivities are traded off against additional qualitative information in the form of potential-based current response.
The sensitivity of voltammetry-based ECD is somewhat improved though square-wave voltammetry (SWV). Hardware particularly adapted employing SWV in electrochemical detection of analytes in a CE separated stream is described in Gerhardt et al., xe2x80x9cSquare-Wave Voltammetry for Capillary Electrophoresis,xe2x80x9d 1998 Anal. Chem. 70, pp. 2167-2173.
However, even with the newly developed CE-SWV system, detection of some analytes was still not possible.
In accordance with one aspect of the invention, a method is provided for electrochemical detection of an analyte in a fluid. The method includes providing a working electrode with a diameter of about 1 xcexcm to 100 xcexcm in association with the fluid. A first positive potential pulse is applied to the fluid at the working electrode at a voltage of less than about 1,000 mV. A negative analytical potential pulse is applied to the fluid at the working electrode at a voltage between about 0 mV and xe2x88x922,000 mV. The current flow through the working electrode is measured during the analytical potential pulse.
In accordance with another aspect of the present invention, an electrochemical detector is provided. The detector includes electronic hardware and software controlling voltage applied to a sample fluid. Voltage is applied in repeated cycles of pulses, where each cycle includes: a positive electrode preparation pulse of a first magnitude; a negative analytical pulse; and a positive cleaning pulse of a second magnitude greater than the first magnitude.
The methods and apparatus described herein permit electrochemical detection of analytes previously thought to be electrochemically inactive. Using these methods and apparatus, analytes produce a positive current flow and a good detection sensitivity.
These and other aspects of the invention will be readily apparent to the skilled artisan in view of the disclosure herein and the appended claims.